PROJECT 3 - PROJECT SUMMARY Macrophages are the most abundant innate immune cells in tissues where they function as immune sentinels. They are uniquely equipped to sense and respond to microbial injury1-4 playing a key role in tissue responses to host pathogens. Emerging studies from our lab, and others, have started to reveal that, counter to earlier dogma, macrophages from different tissues are distinct in their phenotype, function and molecular architecture, and this has important implications for how different tissues respond to infections. Most of our understanding of human macrophage biology, including how macrophages react and respond to pathogens and adjuvants, is based on monocyte-derived macrophages generated in vitro in pre-defined culture conditions. It is now clear that in vitro generated macrophages are different in phenotype and function from actual tissue-resident macrophages, and thus our comprehension of human macrophages is lacking and even inaccurate in many ways. For example, we do not know which TLRs are expressed by different macrophages, which clouds our understanding of pathogen and adjuvant responses in various tissues. Macrophages are the first targets of CMV infection, and are crucial for CMV persistence and dissemination. Since macrophages are also a key modulator of the immune response, these cells are at the crossroad between protection and viral pathogenesis. However to date, human macrophage?s response to CMV and the macrophage-specific contribution to the immune response against CMV is poorly defined. Our goal is to fill this gap in knowledge and understand how different human tissue-resident macrophages respond to pathogens, and to broadly determine the underlying programs that control human tissue macrophage biology. Based on the emerging findings from mice and preliminary studies with human samples, we hypothesize that human macrophages are defined by their microenvironment, which results in molecularly and functionally distinct macrophage populations in different tissues, which have unique responses to pathogens, including CMV. The limited availability of healthy human tissue-resident macrophages has made it impossible to test this hypothesis in humans. To overcome this limitation, we have teamed up with the Farber lab, who has established a truly unique pipeline for obtaining primary human cells from different tissues of organ donors. As part of the HIPC, we will isolate macrophages from different human organs and (1) define the specific properties of macrophages in lymphoid and mucosal tissues, (2) identify tissue-specific macrophage response to innate stimuli, and (3) determine the influence of CMV infection on tissue-resident macrophage function. The outcome of these studies will provide a deep characterization of human tissue-resident macrophages across and between individuals, which will also serve as an important hypothesis-generating resource for the community, help us understand the innate response of different tissues, which can aid vaccine design, and determine how a chronic CMV infection impacts the biology of tissue-resident human macrophages.